SULPHUR AND NITROGEN BACTERIA 225 



that many organisms are injured by carbonate of ammonia, even in minute 

 doses. At all events, any further elaboration of the ammonia is unknown and 

 improbable, more especially it must not be assumed that ammonia arises from 

 the urea in the same way as it does from a pure peptone nutrient. Since 

 urea does not act as a source of carbon to uro-bacteria they cannot thrive on 

 it alone (BEIJERINCK, 1901) ; it supplies the nitrogen need only. As far as their 

 carbon requirements are concerned the individual forms behave very differently; 

 species which get on in acetic or oxalic acid are least exacting, but these form 

 only small quantities of carbonate of ammonia ; those which grow in tartaric 

 acid produce larger quantities, and those which live in malic acid still more. 

 The greatest amount of ammonia is formed by Urobacillus pasteurii and 

 Urococcus ureae, which use bouillon as a source of carbon, for in a thin sowing 

 they completely transform 10-12 g. of urea in 100 g. of fluid in the course of 

 a few days. The immediate factor in the formation of ammonia is an enzyme, 

 urease, as to whose occurrence or absence many contradictory statements have 

 been made. As the result of BEIJERINCK'S recent work, its existence can no longer 

 be doubted ; for this author was able to prove that uro-bacteria killed by the 

 action of chloroform acted as effectively,on urea as living forms. He also estab- 

 lished the fact that the urease cannot diffuse out of the cells, so that previous state- 

 ments as to the solubility of urease are to be explained by assuming that some 

 minute Bacteria in the fluid had been overlooked. [MiQUEL (1904) holds that the 

 view taken as to the solubility of urease is correct.] Ammonia also arises 

 from uric acid ; we need not trace the fate of hippuric acid at present. 



Ammonia, in whatever way it arises, is, as we have already said, absorbed 

 and retained in the soil, and undergoes, as we shall see, change into nitrous 

 and nitric acids. This process, known as nitrification, taking place everywhere 

 in arable soil, was previously considered as a simple oxidation due to inorganic 

 agencies. The observations of SCHLOSSING and MUNTZ (1877-1879) on the 

 dependence of nitrification on external conditions, and especially the effect 

 of temperature and anaesthetics on them, could only be explained by assum- 

 ing the action of lower organisms. From such a conception of the process 

 to the certain isolation of the operative Bacteria was a further and more diffi- 

 cult step. Several authors attempted, with the aid of the usual bacteriologi- 

 cal methods (nutrient gelatine), to isolate the nitro-bacteria from the soil, 

 and not infrequently they succeeded in obtaining pure cultures, to which 

 they ascribed the power of inducing nitrification. But nitrification took place 

 within such circumscribed limits that it was impossible to avoid the suspicion 

 that the nitrate reaction obtained from the cultures was due less to the activity 

 of Bacteria than to the absorption of nitrates from the air. Nitrates occur 

 abundantly in the air, especially of laboratories, and it is well known that 

 nitrates are vigorously absorbed by alkaline fluids (BAUMANN, 1888). 



The labours of WARINGTON (1888) and FRANKLAND (1889) have resulted 

 in an advance in our knowledge of this subject ; but it is due to WINO- 

 GRADSKY (1890, 1891) that the physiology of the nitro-bacteria has been 

 ultimately cleared up, and his work has the right to be considered as 

 one of the most important discoveries in physiology. It is essentially WINO- 

 GRADSKY'S description that we shall follow in our account of the phenomenon. 



[WlNOGRADSKY, 1904.] 



His experiences in dealing with the sulphur- and iron-bacteria had trained 

 this investigator perfectly for the study of the nitro-bacteria. He^had in those 

 cases to deal with highly characteristic types of organism, distinguished from 

 the mass of Bacteria by their varying behaviour to organic food materials. 

 The methods of isolation and culture of technical bacteriology which he had 

 employed in his experiments had completely failed ; might not these failures in 

 the case of the nitro-bacteria be explained by the fact that these organisms require 



JOST Q 



